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CN117402813B - 一种猫肾crfk贴壁细胞系的建立及悬浮驯化与应用 - Google Patents

一种猫肾crfk贴壁细胞系的建立及悬浮驯化与应用 Download PDF

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CN117402813B
CN117402813B CN202311356741.9A CN202311356741A CN117402813B CN 117402813 B CN117402813 B CN 117402813B CN 202311356741 A CN202311356741 A CN 202311356741A CN 117402813 B CN117402813 B CN 117402813B
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crfk
cell line
virus
cells
feline
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CN117402813A (zh
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石文达
张中华
董昊
赵晓
纪志辉
刘继红
王宏伟
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Taizhou Bolai Deli Biotechnology Co ltd
Zhongruidongjian Beijing Biotechnology Co ltd
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Zhongruidongjian Beijing Biotechnology Co ltd
Taizhou Bolai Deli Biotechnology Co ltd
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Abstract

本发明提供一种猫肾CRFK贴壁细胞系的建立及悬浮驯化与应用。所述猫肾细胞系，命名为CRFK‑BLA，保藏在中国微生物菌种保藏管理委员会普通微生物中心，地址：北京市朝阳区北辰西路1号院3号中国科学院微生物研究所，保藏编号为CGMCC NO：45703，分类命名为：猫肾细胞，保藏日期为：2023年8月17日。在其基础上驯化获得的无血清全悬浮培养型CRFK细胞系，命名为CRFK‑BLB，保藏在中国微生物菌种保藏管理委员会普通微生物中心，地址：北京市朝阳区北辰西路1号院3号中国科学院微生物研究所，保藏编号为CGMCC NO：45704，分类命名为：猫肾悬浮细胞，保藏日期为：2023年8月17日。上述细胞形态以均一透亮圆形为主；病毒敏感性试验显示，该猫肾悬浮细胞系对猫细小病毒、犬细小病毒、猫泛白细胞减少症病毒、猫杯状病毒、猫传染性鼻气管炎病毒和水貂病毒性肠炎病毒敏感；该细胞系可用于培养、分离、检测病毒，制备病毒疫苗，筛选用于预防或治疗病毒感染所致的疾病的药物。

Description

一种猫肾CRFK贴壁细胞系的建立及悬浮驯化与应用
技术领域
本发明属于生物技术领域，具体涉及一种猫肾CRFK贴壁细胞系的建立及悬浮驯化与应用。
背景技术
随着宠物行业的不断发展，宠物饲养数量逐年增多，以宠物猫和宠物狗为主，在宠物带给人们快乐的同时，宠物健康问题也威胁着公共安全。目前国内动物疫苗企业商品化的宠物疫苗不多，质量参差不齐，主要依赖进口，产品价格昂贵，国内很多单位正着手这一领域产品的开发。目前危害宠物猫的病毒主要有猫细小病毒，犬细小病毒，猫冠状病毒，猫疱疹病毒等，而对宠物猫产生严重危害的病毒就是猫细小病毒。
传统的CRFK细胞培养多采用有血清贴壁培养方式。血清是由血浆去除纤维蛋白而形成的一种复杂混合物，其含有细胞生长所需的生长因子、激素、载体蛋白、贴壁因子、微量元素以及其他营养物质，可有效地促进细胞生长和产物表达。然而，血清的应用也存在许多问题：易受病毒、支原体或其他病原体的污染；批间差异造成产品批次间的质量难以严格控制；大量血清蛋白的存在增加了下游分离纯化的难度，部分蛋白难以通过分离纯化手段彻底去除，影响了产品的最终质量；此外，血清来源困难、价格昂贵，大规模动物细胞培养过程中使用血清会增加生产成本。CRFK和F81均属于上皮样猫肾细胞系。CRFK最早在1964年由Crandell分离，F81是由CRFK通过传代克隆筛选转化而来的工程细胞系。两种细胞均易于贴壁培养，对多种宠物病毒和特产经济动物病毒较敏感，在兽医生物制品研究中得到了广泛应用。如犬细小病毒(CPV)、猫泛白细胞减少症病毒(FPV)、猫疱疹病毒(FHV)、猫杯状病毒(FCV)、水貂病毒性肠炎病毒(MEV)等。
鉴于猫病毒性抗原(如FPV、FHV、FCV)在常规CRFK和F81细胞系上敏感性不一致，无法用单一细胞系生产多联宠物疫苗。为方便实验室研究和工艺开发，进行猫肾原代细胞的分离与传代细胞系的建系研究；分选出对宠物病毒敏感性更强的CRFK细胞系。CRFK细胞的培养方法基本采用二维单细胞层贴壁培养。在单细胞层培养体系中，细胞增殖易受到基质表面积的限制，难以实现大规模生产，且消化过程亦会增加工艺的复杂性、生产时间和成本。而单细胞悬浮培养可以不受细胞生长表面的限制，易于实现细胞及产品的大规模生产。有关CRFK细胞培养方式、代谢特征和培养过程的研究工作不多。因此，CRFK细胞无血清单细胞悬浮培养系统的开发与研究对其产业化进程意义重大。
发明内容
为了解决上述技术难题，本研究选取10日龄无特异性病原体幼猫肾脏，经消化研磨后于含10％胎牛血清的DMEM培养基进行培养，经连续筛选和传代验证细胞传代稳定性，经外源病毒、支原体检验后建立CRFK细胞系种子库。结果表明，分离的猫肾原代细胞经连续10代筛选培养后，细胞形态基本一致；连续传代至45代，细胞形态单一且生长速率稳定、外源病毒及支原体检验合格；成功建立猫肾细胞传代细胞系，命名为CRFK，呈上皮细胞样，属自发永生细胞系。而且在此基础上，利用无血清培养基中成功进行了无血清驯化，并通过单细胞悬浮培养驯化方法，获得了适合无血清单细胞悬浮培养的CRFK-S细胞株。为CRFK细胞培养、病毒疫苗扩增的过程优化和放大奠定了基础，同时也为其他动物细胞培养过程和疫苗等生物制品的工业化生产提供借鉴。
具体的本发明提供一种猫肾细胞系，命名为CRFK-BLA，保藏在中国微生物菌种保藏管理委员会普通微生物中心，地址：北京市朝阳区北辰西路1号院3号中国科学院微生物研究所，保藏编号为CGMCC NO：45703，分类命名为：猫肾细胞，保藏日期为：2023年8月17日。
进一步的，本发明提供一种CRFK细胞的无血清驯化方法，包括如下步骤；
CRFK细胞准备将复苏的CRFK细胞(CRFK-BLA保藏株)接种到含8％胎牛血清的DMEM培养基中，按照常规方法传代培养5代；
CRFK细胞梯度降血清培养将上述所获得的贴壁培养型CRFK细胞依次用含6％、4％、2％胎牛血清的DMEM培养基各培养6代，按1∶3比例进行传代培养，于37℃、含5％CO2细胞培养箱中培养；
低血清全悬浮培养基CRFK细胞贴壁培养用含2％胎牛血清的全悬浮培养基，将上步骤中最后一代所获得的细胞进行贴壁培养；待细胞贴壁、培养瓶中单层汇合度达90％以上后，弃去培养液；用0.125％ EDTA-胰酶消化分散，按1∶2比例进行传代培养，于37℃、含5％CO2细胞培养箱中培养。连续传代培养5代，得到适应全悬浮培养基低血清贴壁培养型CRFK细胞系。
低血清全悬浮培养基CRFK细胞悬浮培养用含2％胎牛血清的全悬浮培养基将上步骤中最后一代所获细胞密度调整至1×106cells/mL，于37℃、含5％CO2，120r/min摇床培养；待细胞密度达到4×106cells/mL时，用含1％胎牛血清的全悬浮培养基按1∶4比例分瓶培养；待细胞密度达到4×106cells/mL时，用含0.5％胎牛血清的全悬浮培养基按1∶3比例分瓶培养；连续传代五次后，驯化得到低血清全悬浮培养型CRFK细胞系，于37℃、含5％CO2，120r/min摇床培养，驯化得到低血清全悬浮培养型CRFK细胞系。
进一步的，本发明提供一种CRFK单细胞悬浮无血清驯化方法，所述方法包括步骤：
96孔板筛选单细胞将低血清全悬浮培养型CRFK细胞系，离心后加入无血清全悬浮培养基，吹打悬浮细胞并计数，将细胞用无血清全悬浮培养基稀释，制成10cells/mL的细胞悬液，按100μl/孔加入96孔细胞培养板，于37℃、含5％CO2细胞培养箱中培养。
24孔板筛选单细胞培养48～72小时后，从96孔细胞培养板中选数个仅有1个细胞/孔的培养孔，挑选出生长良好的单细胞株；将其转移至24孔板，于37℃、含5％CO2细胞培养箱中培养。
6孔板筛选单细胞培养48～72小时后，从24孔细胞培养板中挑选出生长良好的单细胞株；将其转移至6孔板，于37℃、含5％CO2细胞培养箱中培养。
单细胞贴壁放大培养培养48～72小时后，从6孔细胞培养板中挑选出生长良好的单细胞株；将其转移至25cm2培养瓶，于37℃、含5％CO2细胞培养箱中培养。
单细胞悬浮培养待细胞生长至90％时，按上述方法消化后，转移至摇瓶继续培养。调整细胞密度为5×105cells/mL，于37℃、含5％CO2，120r/min摇床培养，驯化得到无血清全悬浮培养型CRFK细胞系。
进一步的，本发明提供一种无血清全悬浮培养型CRFK细胞系，命名为CRFK-BLS，保藏在中国微生物菌种保藏管理委员会普通微生物中心，地址：北京市朝阳区北辰西路1号院3号中国科学院微生物研究所，保藏编号为CGMCC NO：45704，分类命名为：猫肾悬浮细胞，保藏日期为：2023年8月17日。
进一步的，本发明提供一种上述猫肾细胞系和/或无血清全悬浮培养型CRFK细胞系在(1)～(8)中任一项中的应用；
(1)培养病毒；
(2)制备培养病毒的产品；
(3)分离病毒；
(4)制备分离病毒的产品；
(5)非诊断治疗目的地检测病毒；
(6)制备检测病毒的产品；
(7)制备病毒疫苗；
(8)药物筛选。
所述病毒为犬细小病毒、猫泛白细胞减少症病毒、猫杯状病毒、猫传染性鼻气管炎病毒和水貂病毒性肠炎病毒；
所述药物为用于预防或治疗病毒感染所致的疾病的药物。
利用上述猫肾细胞系和/或无血清全悬浮培养型CRFK细胞系培养病毒疫苗的方法。
附图说明
图1是猫肾原代细胞F10代(20×)。
图2是CRFK细胞系不同代次(20×)，其中A为CRFK-F15(15代)；B为CRFK-F20(20代)。图3是CRFK细胞系病毒培养敏感性实验(20×)，其中，A为CRFK-BLA对照，B为CRFK-BLA-FPV，C为CRFK-BLA-FHV；D为CRFK对照,E为CRFK-FPV，F为CRFK-FHV。
图4CRFK和CRFK-BLA的病毒滴度。
图5是CRFK细胞系低血清及悬浮驯化结果(20×)。A是CRFK-贴壁在2％FBS+DMEM中的贴壁生长，B是CRFK-悬浮在0.5％FBS+全悬浮培养基中悬浮生长。
图6是CRFK细胞单克隆细胞株生长过程(20×)；
图7悬浮CRFK-R细胞系镜下结果(20×)；
图8悬浮CRFK-R细胞系镜生长规律。
图9CRFK-R细胞系的病毒敏感性实验。
具体实施方式
以下是本发明具体的实施方案，以进一步阐述本发明，但不能解释为限制本发明的范围。本领域技术人应该理解的是，在不偏离本发明的精神和范围下可以对本发明技术方案的细节和形式进行修改或替换，但这些修改或替换均落入本发明的保护范围内。
下述实施例中所使用的实验方法如无特殊说明，均为常规方法。
实验涉及的主要生物材料和设备
毒种猫细小病毒HBX05株、猫疱疹病毒BJS01株和猫杯状病毒BJH13株，由北京博莱得利生物技术有限责任公司分离、鉴定并保存。
实验动物10日龄无特性异性病原体雌性幼猫(FPV、FHV、FCV抗原检测阴性)。
主要试剂病毒基因组DNA/RNA提取试剂盒、RNA反转录试剂盒，购自北京全式金生物技术有限公司；PCR和RT-PCR扩增试剂盒，购自宝生物工程(大连)有限公司；硫乙醇酸盐流体培养基(TG)、胰酪大豆胨液体培养基(TSB)和支原体液体培养基、支原体固体培养基，购自北京中海生物科技有限公司；高糖DMEM培养基(ME105-001)、特级南美胎牛血清(S711-001S)，购自苏州双汝生物科技有限公司。
无血清培养基在DMEM培养基的基础上，添加胰岛素、转铁蛋白、胆固醇、碳酸氢钠、HEPES、Pluronic F68等成分配制而成(具体成分见下表)，经0.1μm微孔滤膜过滤除菌。
实施例1：CRFK细胞系建立
猫肾原代细胞的分离与培养 将筛选的无特性异性病原体幼猫无痛处死后，浸泡于75％酒精溶液中；于生物安全柜剖开腹腔取出肾脏，剥离肾组织筋膜层；取肾脏于无菌剪刀剪碎至米粒大小，用无菌PBS缓冲液冲洗3次；加入20ml浓度为0.5mg/ml胰蛋白酶，置于37℃恒温培养箱中消化1小时；用无菌研磨器研磨后，使用100目筛网过滤细胞悬液；于700r/min离心5分钟，用完全培养基(含10％胎牛血清的DMEM)清洗三次；于10ml完全培养基重悬后，置于37℃，含5％ CO2恒温培养箱中培养，记作F1。
待原代细胞培养24小时后，于预热的PBS清洗3次后更换新的完全培养基；长满单层后，用0.125％的胰酶冲洗细胞，冲洗掉状态不好的细胞；弃液后，重新加入0.125％的胰酶进行消化；显微镜观察细胞分散为单个细胞后立即加入完全培养基，按1∶3进行传代。按此方式培养至F10。经连续10代分离培养后，猫肾原代细胞状态良好。取F10代长至良好单层的猫肾原代细胞于显微镜下观察，细胞呈菱形、长椭圆形、轮廓清晰(见图1)
取状态良好的单层原代猫肾细胞，用0.125％的胰酶冲洗细胞；弃液后，重新加入0.125％的胰酶进行消化；显微镜观察细胞分散为单个细胞后立即加入完全培养基，按1∶3进行传代；于37℃、含5％CO2细胞培养箱中培养。按上述方法连续传代至F45，观察细胞形态及状态。各代次细胞形态正常，呈菱形、长椭圆形、轮廓清晰(见图2)；细胞生长速率稳定。说明猫肾细胞原代细胞经连续筛选培养后可作为传代细胞系，以CRFK命名，为自发永久细胞系。
取状态良好的单层原代猫肾细胞，用0.125％的胰酶冲洗细胞；弃液后，重新加入0.125％的胰酶进行消化；显微镜观察细胞分散为单个细胞后立即加入完全培养基制成细胞悬液，计数；800r/min离心8～10分钟，弃上清，加入冻存液(DMEM∶胎牛血清∶DMSO＝6∶3∶1)轻轻吹打混匀后，调整细胞浓度为2.0×106/ml，移入冻存管中，每管1.0ml，标记后于液氮中保存备用。
CRFK细胞系纯净性检验
细胞复苏将冻存于液氮中的CRFK细胞取出，置于37℃水浴迅速融化，800r/min离心8～10分钟。加入完全血清培养基，轻轻吹打重悬细胞后移入T25细胞瓶中；于37℃、含5％CO2细胞培养箱中培养。
无菌检验 取CRFK细胞，按2020版《中国兽药典》三部附录3306进行检验。
支原体检验 取CRFK细胞，按2020版《中国兽药典》三部附录3308进行检验。
外源病毒检验 取CRFK细胞，按2020版《中国兽药典》三部附录3305进行检验。
对CRFK细胞库细胞F10、F20、F30复苏后进行纯净性检验，各代词细胞均检验合格，无细菌、支原体及外源病毒污染。
将第F20分离纯化扩增后，命名为CRFK-BLA，保藏在中国微生物菌种保藏管理委员会普通微生物中心，地址：北京市朝阳区北辰西路1号院3号中国科学院微生物研究所，保藏编号为CGMCC NO：45703，分类命名为：猫肾细胞，保藏日期为：2023年8月17日。
实施例2：CRFK细胞系病毒敏感性检验
取猫细小病毒(HBX05株)、猫疱疹病毒(BJS01株)和猫杯状病毒(BJH13株)按体积比分别接种于T25细胞瓶的CRFK细胞和CRFK-BLA保藏株细胞；(①FPV：同步接种，按5％比例接种于10ml含2％牛血清的CRFK细胞悬液；②FHV：按0.1％接种于已长成单层的CRFK细胞上；③FCV：按0.1％接种于已长成单层的CRFK细胞上)；同时设正常细胞对照；置37℃、含5％CO2培养箱中培养观察。观察CRFK-BLA细胞系是否对各毒株敏感；当细胞病变80％时，收集病毒液，冻融3次后测定病毒TCID50。结果显示，CRFK-BLA细胞接毒后可见明显细胞病变(见图3)。FPV HBX05株同步接种后镜下可观察到细胞出现肿胀变圆、拉丝等典型的病变；FHVBJS01株接种后镜下可观察到细胞呈圆缩、脱落病变；将FCV BJH13株接种后镜下可观察到细胞聚缩成团、脱落、葡萄串样典型病变。CRFK-BLA所培养的病毒，病毒滴度均高于CRFK细胞(图4)。
实施例3：CRFK细胞系的无血清驯化
采用逐步适应法梯度降低培养基中血清含量。
CRFK细胞准备将复苏的CRFK细胞(CRFK-BLA保藏株)接种到含8％胎牛血清的DMEM培养基中，以DMEM为基础培养基，添加8％胎牛血清进行培养(pH值为6.8～7.2)；于37℃、含5％CO2细胞培养箱中培养。待细胞贴壁、培养瓶中单层汇合度达90％以上后，弃去培养液，用0.125％的EDTA-胰酶消化分散，按照常规方法传代培养5代。
CRFK细胞梯度降血清培养将上述所获得的贴壁培养型CRFK细胞依次用含6％、4％、2％胎牛血清的DMEM培养基各培养6代，按1∶3比例进行传代培养，于37℃、含5％CO2细胞培养箱中培养。
低血清全悬浮培养基CRFK细胞贴壁培养用含2％胎牛血清的全悬浮培养基(如前述配方)，将上步骤中最后一代所获得的细胞进行贴壁培养；待细胞贴壁、培养瓶中单层汇合度达90％以上后，弃去培养液；用0.125％ EDTA-胰酶消化分散，按1∶2比例进行传代培养，于37℃、含5％CO2细胞培养箱中培养。连续传代培养5代，得到适应全悬浮培养基低血清贴壁培养型CRFK细胞系。
低血清全悬浮培养基CRFK细胞悬浮培养用含2％胎牛血清的全悬浮培养基将上步骤中最后一代所获细胞密度调整至1×106cells/mL，于37℃、含5％CO2，120r/min摇床培养；待细胞密度达到4×106cells/mL时，用含1％胎牛血清的全悬浮培养基按1∶4比例分瓶培养；待细胞密度达到4×106cells/mL时，用含0.5％胎牛血清的全悬浮培养基按1∶3比例分瓶培养；连续传代五次后，驯化得到低血清全悬浮培养型CRFK细胞系，于37℃、含5％CO2，120r/min摇床培养。
经连续梯度降血清后，该自分离CRFK细胞系可在含2％胎牛血清的DMEM培养基中正常贴壁生长与传代(见图5A)；细胞形态不变，呈菱形、长椭圆形、轮廓清晰。经低血清全悬浮培养基悬浮驯化后，CRFK细胞系可在含0.5％胎牛血清的全悬浮培养基中正常生长(见图5B)；镜下观察呈单个、透亮、细胞轮廓清晰，透亮度高。表明驯化得到低血清全悬浮培养型CRFK细胞系。
实施例4：CRFK单细胞悬浮驯化方法
96孔板筛选单细胞将低血清全悬浮培养型CRFK细胞系，离心后加入无血清全悬浮培养基(如前述配方)，吹打悬浮细胞并计数，将细胞用无血清全悬浮培养基稀释，制成10cells/mL的细胞悬液，按100μl/孔加入96孔细胞培养板，于37℃、含5％CO2细胞培养箱中培养。
24孔板筛选单细胞培养48～72小时后，从96孔细胞培养板中选数个仅有1个细胞/孔的培养孔，挑选出生长良好的单细胞株；将其转移至24孔板，于37℃、含5％CO2细胞培养箱中培养。
6孔板筛选单细胞培养48～72小时后，从24孔细胞培养板中挑选出生长良好的单细胞株；将其转移至6孔板，于37℃、含5％CO2细胞培养箱中培养。
单细胞贴壁放大培养培养48～72小时后，从6孔细胞培养板中挑选出生长良好的单细胞株；将其转移至25cm2培养瓶，于37℃、含5％CO2细胞培养箱中培养。
单细胞悬浮培养待细胞生长至90％时，按上述方法消化后，转移至摇瓶继续培养。调整细胞密度为5×105cells/mL，于37℃、含5％CO2，120r/min摇床培养，驯化得到无血清全悬浮培养型CRFK细胞系(见图6)；该细胞株可在无血清培养基中正常生长，细胞形态不变，呈菱形、长椭圆形、轮廓清晰。转至摇瓶培养后，按5×105cells/mL传代培养，以无血清培养基中连续传代培养5代，CRFK细胞系全悬浮培养基中正常悬浮生长(见图7)；镜下观察呈单个、透亮、细胞轮廓清晰，透亮度高。命名为CRFK-BLB。
将上述驯化得到无血清全悬浮培养型CRFK细胞分离纯化扩增后，命名为CRFK-BLB，保藏在中国微生物菌种保藏管理委员会普通微生物中心，地址：北京市朝阳区北辰西路1号院3号中国科学院微生物研究所，保藏编号为CGMCC NO：45704，分类命名为：猫肾悬浮细胞，保藏日期为：2023年8月17日。
将建立的CRFK-BLB细胞系进行传代，按1×106cells/mL传代培养，每24h以台盼蓝染色，用血球计数板计死细胞和活细胞数，计算细胞活率结果显示(见图8)，悬浮培养96h后细胞达到最大密度4.5×106cell/mL，活率为90±0.5％，倍增时间在25.37±0.35小时。表明该单克隆细胞株(CRFK-BLB)生长稳定性较好。
病毒敏感性试验将建立的CRFK-BLB细胞系进行传代，按1×106cells/mL传代培养，当细胞密度至2.0×106cells/mL时，取猫细小病毒(HBX05株)、猫疱疹病毒(BJS01株)和猫杯状病毒(BJH13株)按体积比接种于CRFK悬浮培养摇瓶(①FPV：按5％比例接种；②FHV：按0.1％接种；③FCV：按0.1％接种)；同时设正常细胞对照；于37℃、含5％CO2，120r/min摇床培养。观察该CRFK-R细胞系是否对各毒株敏感。收集病毒液，冻融3次后测定病毒TCID50(见图9)。证实该单克隆细胞株(CRFK-BLB)较传统贴壁CRFK细胞更利于FPV及FHV培养。
上述对实施例的描述是为了便于该技术领域的普通技术人员能理解和使用本发明。熟悉本领域技术人员显然可以容易的对这些实施例做出各种修改，并把在此说明的一般原理应用到其他实施例中，而不必经过创造性的劳动。因此，本发明不限于上述实施例。本领域技术人员根据本发明的原理，不脱离本发明的范畴所做出的改进和修改都应该在本发明的保护范围之内。

Claims (4)

1.一种猫肾细胞系，其特征在于所述猫肾细胞系命名为CRFK-BLA，保藏在中国微生物菌种保藏管理委员会普通微生物中心，地址：北京市朝阳区北辰西路1号院3号 中国科学院微生物研究所，保藏编号为CGMCC NO：45703，分类命名为：猫肾细胞，保藏日期为：2023年8月17日。
2.一种无血清全悬浮培养型CRFK细胞系，其特征在于所述无血清全悬浮培养型CRFK细胞系命名为CRFK-BLS，保藏在中国微生物菌种保藏管理委员会普通微生物中心，地址：北京市朝阳区北辰西路1号院3号 中国科学院微生物研究所，保藏编号为CGMCC NO：45704，分类命名为：猫肾悬浮细胞，保藏日期为：2023年8月17日。
3.一种权利要求1所述猫肾细胞系和/或权利要求2所述无血清全悬浮培养型CRFK细胞系在(1)～(8)中任一项中的非疾病诊断目的应用，所述应用为；
(1)培养病毒；
(2)制备培养病毒的产品；
(3)分离病毒；
(4)制备分离病毒的产品；
(5)非诊断治疗目的地检测病毒；
(6)制备检测病毒的产品；
(7)制备病毒疫苗；
(8)药物筛选，
所述病毒为犬细小病毒、猫泛白细胞减少症病毒、猫杯状病毒、猫传染性鼻气管炎病毒和水貂病毒性肠炎病毒。
4.利用权利要求1所述猫肾细胞系和/或权利要求2所述无血清全悬浮培养型CRFK细胞系培养病毒疫苗的方法，所述病毒为犬细小病毒、猫泛白细胞减少症病毒、猫杯状病毒、猫传染性鼻气管炎病毒和水貂病毒性肠炎病毒。
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